Persistent epigenetic signals propel a senescence-associated secretory phenotype and trained innate immunity in CD34+ hematopoietic stem cells from diabetic patients.

BACKGROUND: Diabetes-induced trained immunity contributes to the development of atherosclerosis and its complications. This study aimed to investigate in humans whether epigenetic signals involved in immune cell activation and inflammation are initiated in hematopoietic stem/progenitor cells (HSPCs) and transferred to differentiated progeny. METHODS AND RESULTS: High glucose (HG)-exposure of cord blood (CB)-derived HSPCs induced a senescent-associated secretory phenotype (SASP) characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27genes, upregulation of NFkB-p65 transcription factor and increased secretion of the inflammatory cytokines TNFα and IL6. Chromatin immunoprecipitation assay (ChIP) of p65 promoter revealed that H3K4me1 histone mark accumulation and methyltransferase SetD7 recruitment, along with the reduction of repressive H3K9me3 histone modification, were involved in NFkB-p65 upregulation of HG-HSPCs, as confirmed by increased RNA polymerase II engagement at gene level. The differentiation of HG-HSPCs into myeloid cells generated highly responsive monocytes, mainly composed of intermediate subsets (CD14hiCD16+), that like the cells from which they derive, were characterized by SASP features and similar epigenetic patterns at the p65 promoter. The clinical relevance of our findings was confirmed in sternal BM-derived HSPCs of T2DM patients. In line with our in vitro model, T2DM HSPCs were characterized by SASP profile and SETD7 upregulation. Additionally, they generated, after myeloid differentiation, senescent monocytes mainly composed of proinflammatory intermediates (CD14hiCD16+) characterized by H3K4me1 accumulation at NFkB-p65 promoter. CONCLUSIONS: Hyperglycemia induces marked chromatin modifications in HSPCs, which, once transmitted to the cell progeny, contributes to persistent and pathogenic changes in immune cell function and composition.

Circulating levels of AGEs and soluble RAGE isoforms are associated with all-cause mortality and development of cardiovascular complications in type 2 diabetes: a retrospective cohort study.

BACKGROUND: Advanced glycation end-products (AGEs) and their interaction with the receptor for advanced glycation end-products (RAGE) play a pivotal role in the development and progression of type 2 diabetes. In this retrospective cohort study, we explored the association of circulating levels of soluble RAGE (sRAGE) isoforms, i.e., endogenous secretory esRAGE and cleaved cRAGE, AGEs and their respective ratios with 15-year all-cause mortality in type 2 diabetes. METHODS: Baseline AGEs and sRAGE isoforms concentration were measured by ELISA in 362 patients with type 2 diabetes and in 125 age- and gender-matched healthy control subjects (CTR). Independent predictors of mortality were determined using Cox proportional-hazards models and used to build and validate a nomogram for all-cause mortality prediction in type 2 diabetes. RESULTS: AGEs, total sRAGE, cRAGE and the AGEs/sRAGE and AGEs/esRAGE ratios were significantly increased in patients with type 2 diabetes compared to CTR (p < 0.001). In CTR subjects, but not in type 2 diabetes patients, a significant negative correlation between cRAGE and age was confirmed (p = 0.003), whereas the AGEs/sRAGE (p = 0.032) and AGEs/cRAGE (p = 0.006) ratios were positively associated with age. At an average follow-up of 15 years (4,982 person-years), 130 deaths were observed. The increase in the AGEs/cRAGE ratio was accompanied by a higher risk of all-cause mortality in patients with type 2 diabetes (HR per each SD increment = 1.30, 95% CI 1.15-1.47; p < 0.001). Moreover, sRAGE was associated with the development of major adverse cardiovascular events (MACE) in type 2 diabetes patients without previous MACE (OR for each SD increase: 1.48, 95% CI 1.11-1.89). A nomogram based on age, sex, HbA1c, systolic blood pressure, and the AGEs/cRAGE ratio was built to predict 5-, 10- and 15-year survival in type 2 diabetes. Patients were categorized into quartiles of the monogram scores and Kaplan-Meier survival curves confirmed the prognostic accuracy of the model (log-rank p = 6.5 × 10- 13). CONCLUSIONS: The ratio between AGEs and the cRAGE isoform is predictive of 15-year survival in patients with type 2 diabetes. Our data support the assessment of circulating AGEs and soluble RAGE isoforms in patients with type 2 diabetes as predictors of MACE and all-cause mortality.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure.

BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

MicroRNA-34a: the bad guy in age-related vascular diseases.

The age-related vasculature alteration is the prominent risk factor for vascular diseases (VD), namely, atherosclerosis, abdominal aortic aneurysm, vascular calcification (VC) and pulmonary arterial hypertension (PAH). The chronic sterile low-grade inflammation state, alias inflammaging, characterizes elderly people and participates in VD development. MicroRNA34-a (miR-34a) is emerging as an important mediator of inflammaging and VD. miR-34a increases with aging in vessels and induces senescence and the acquisition of the senescence-associated secretory phenotype (SASP) in vascular smooth muscle (VSMCs) and endothelial (ECs) cells. Similarly, other VD risk factors, including dyslipidemia, hyperglycemia and hypertension, modify miR-34a expression to promote vascular senescence and inflammation. miR-34a upregulation causes endothelial dysfunction by affecting ECs nitric oxide bioavailability, adhesion molecules expression and inflammatory cells recruitment. miR-34a-induced senescence facilitates VSMCs osteoblastic switch and VC development in hyperphosphatemia conditions. Conversely, atherogenic and hypoxic stimuli downregulate miR-34a levels and promote VSMCs proliferation and migration during atherosclerosis and PAH. MiR34a genetic ablation or miR-34a inhibition by anti-miR-34a molecules in different experimental models of VD reduce vascular inflammation, senescence and apoptosis through sirtuin 1 Notch1, and B-cell lymphoma 2 modulation. Notably, pleiotropic drugs, like statins, liraglutide and metformin, affect miR-34a expression. Finally, human studies report that miR-34a levels associate to atherosclerosis and diabetes and correlate with inflammatory factors during aging. Herein, we comprehensively review the current knowledge about miR-34a-dependent molecular and cellular mechanisms activated by VD risk factors and highlight the diagnostic and therapeutic potential of modulating its expression in order to reduce inflammaging and VD burn and extend healthy lifespan.

Doxorubicin induces an alarmin-like TLR4-dependent autocrine/paracrine action of Nucleophosmin in human cardiac mesenchymal progenitor cells.

BACKGROUND: Doxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs. RESULTS: We found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion. CONCLUSIONS: We hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.

Effects of RAGE Deletion on the Cardiac Transcriptome during Aging.

Cardiac aging is characterized by increased cardiomyocyte hypertrophy, myocardial stiffness, and fibrosis, which enhance cardiovascular risk. The receptor for advanced glycation end-products (RAGE) is involved in several age-related diseases. RAGE knockout (Rage-/-) mice show an acceleration of cardiac dimension changes and interstitial fibrosis with aging. This study identifies the age-associated cardiac gene expression signature induced by RAGE deletion. We analyzed the left ventricle transcriptome of 2.5-(Young), 12-(Middle age, MA), and 21-(Old) months-old female Rage-/- and C57BL/6N (WT) mice. By comparing Young, MA, and Old Rage-/- versus age-matched WT mice, we identified 122, 192, and 12 differently expressed genes, respectively. Functional inference analysis showed that RAGE deletion is associated with: (i) down-regulation of genes involved in antigen processing and presentation of exogenous antigen, adaptive immune response, and cellular responses to interferon beta and gamma in Young animals; (ii) up-regulation of genes related to fatty acid oxidation, cardiac structure remodeling and cellular response to hypoxia in MA mice; (iii) up-regulation of few genes belonging to complement activation and triglyceride biosynthetic process in Old animals. Our findings show that the age-dependent cardiac phenotype of Rage-/- mice is associated with alterations of genes related to adaptive immunity and cardiac stress pathways.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context.

Low influenza vaccination coverage in subjects with liver cirrhosis. An alert waiting for winter season 2020-2021 during the COVID-19 pandemic.

We have evaluated flu vaccine coverage and variables associated with the lack of vaccination in cirrhotic subjects with particular attention to the cirrhosis etiology. Cirrhotic subjects consecutively referring to eight Italian centers were prospectively enrolled for a 6-month period in 2019. Subjects were asked if they had received a flu vaccine in the last 12 months. Multiple logistic regression analysis was performed to identify independent predictors of lack of vaccination. A total of 818 cases were recruited. The overall vaccine coverage was 39.6% (26.9% in those younger than 65 years and 51.9% in those older than 64 years; p < 0.001). Age < 65 years (odds ratio [OR] = 2.38; 95% confidence interval [CI] = 1.68-3.36), alcoholic etiology (OR = 2.40; 95% CI = 1.49-3.85), birth abroad (OR = 2.7; 95% CI = 1.10-6.61), and residence in South/Sardinia island (OR = 1.66; 95% CI = 1.14-2.42) all resulted independent predictors of the likelihood of lack of vaccination. The lack of information regarding the vaccine as the reason for no vaccination was reported by 71.4% of foreigners and by 34.7% of natives (p < 0.001). In conclusion, much work still should be done to improve coverage among groups at higher risk of lack of vaccination identified in this survey. The ongoing SARS-CoV-2 pandemic may represent one more alert for improving seasonal flu vaccine coverage to avoid further stress to the National Health System.

Targeting cancer stem cells in medulloblastoma by inhibiting AMBRA1 dual function in autophagy and STAT3 signalling.

Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

Chronic hepatitis B virus infection in Italy during the twenty-first century: an updated survey in 2019.

The aim of this study is to provide updates on the characteristics of chronic HBsAg carriers in Italy before the advent of new drugs eliminating or functionally inactivating the genome HBV reservoirs. HBV endemicity has greatly decreased in Italy over the past decades. A not negligible number of chronic HBsAg carriers are still alive in the country. Chronic HBsAg carriers consecutively referring to 9 units in Italy were prospectively enrolled for a 6-month period in 2019. Multiple logistic regression analysis was performed to identify independent predictors of treatment. A total of 894 cases was recruited (sex ratio 1.6; mean age 53.7 ± 13.5 years). The proportion of subjects born abroad was 19.0%; only 1% of cases reported current heavy alcohol intake (> 4 units/day). Chronic HBV infection, chronic HBV hepatitis, and subjects with liver cirrhosis and/or HCC represented 24.8%, 55%, and 19.3% of cases, respectively. After exclusion of the 222 subjects with chronic HBV infection, the proportion of subjects under therapy was as high as 89.3%. A more severe liver disease (OR 2.52; 95% CI = 1.25-5.14) resulted an independent predictor of the likelihood of treatment; male sex was marginally associated (OR 1.67; 95% CI = 1.02-2.76) to the chance of treatment. People born abroad had same chance than Italians native to be treated (OR 2.12; 95% CI = 0.9-4.97). The high proportion of subjects under treatment and the absence of gender and ethnic barrier against treatment sound good news. These updated figures may represent reference data for evaluating the potential impact of forthcoming new therapy against HBV-related disease.

Successful use of caplacizumab in a case of refractory acquired thrombotic thrombocytopenic purpura following subacute thyroiditis.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare condition mainly characterized by microangiopathic hemolytic anemia, thrombocytopenia, reported in approximately three cases per one million adults per year. Some reports describing co-occurrence of aTTP and other autoimmune disorders, as Graves' thyroiditis, are reported. To the best of our knowledge this is the first report describing co-occurrence of subacute thyroiditis and aTTP. The patient was refractory to conventional therapy with plasma exchange, steroids and rituximab but was successfully treated with the addition of caplacizumab, an anti-VWF bivalent variable-domain-only immunoglobulin fragment that inhibits interaction between VWF multimers and platelets.

Diabetes Induces a Transcriptional Signature in Bone Marrow-Derived CD34+ Hematopoietic Stem Cells Predictive of Their Progeny Dysfunction.

Hematopoietic stem/progenitor cells (HSPCs) participate in cardiovascular (CV) homeostasis and generate different types of blood cells including lymphoid and myeloid cells. Diabetes mellitus (DM) is characterized by chronic increase of pro-inflammatory mediators, which play an important role in the development of CV disease, and increased susceptibility to infections. Here, we aimed to evaluate the impact of DM on the transcriptional profile of HSPCs derived from bone marrow (BM). Total RNA of BM-derived CD34+ stem cells purified from sternal biopsies of patients undergoing coronary bypass surgery with or without DM (CAD and CAD-DM patients) was sequenced. The results evidenced 10566 expressed genes whose 79% were protein-coding genes, and 21% non-coding RNA. We identified 139 differentially expressed genes (p-value < 0.05 and |log2 FC| > 0.5) between the two comparing groups of CAD and CAD-DM patients. Gene Set Enrichment Analysis (GSEA), based on Gene Ontology biological processes (GO-BP) terms, led to the identification of fourteen overrepresented biological categories in CAD-DM samples. Most of the biological processes were related to lymphocyte activation, chemotaxis, peptidase activity, and innate immune response. Specifically, HSPCs from CAD-DM patients displayed reduced expression of genes coding for proteins regulating antibacterial and antivirus host defense as well as macrophage differentiation and lymphocyte emigration, proliferation, and differentiation. However, within the same biological processes, a consistent number of inflammatory genes coding for chemokines and cytokines were up-regulated. Our findings suggest that DM induces transcriptional alterations in HSPCs, which are potentially responsible of progeny dysfunction.

The Role of Glycemic Variability in Cardiovascular Disorders.

Diabetes mellitus (DM) is one of the most common and costly disorders that affect humans around the world. Recently, clinicians and scientists have focused their studies on the effects of glycemic variability (GV), which is especially associated with cardiovascular diseases. In healthy subjects, glycemia is a very stable parameter, while in poorly controlled DM patients, it oscillates greatly throughout the day and between days. Clinically, GV could be measured by different parameters, but there are no guidelines on standardized assessment. Nonetheless, DM patients with high GV experience worse cardiovascular disease outcomes. In vitro and in vivo studies showed that high GV causes several detrimental effects, such as increased oxidative stress, inflammation, and apoptosis linked to endothelial dysfunction. However, the evidence that treating GV is beneficial is still scanty. Clinical trials aiming to improve the diagnostic and prognostic accuracy of GV measurements correlated with cardiovascular outcomes are needed. The present review aims to evaluate the clinical link between high GV and cardiovascular diseases, taking into account the underlined biological mechanisms. A clear view of this challenge may be useful to standardize the clinical evaluation and to better identify treatments and strategies to counteract this DM aspect.

Migratory flow and hepatitis delta infection in Italy: A new challenge at the beginning of the third millennium.

In Italy, HDV infection endemicity has greatly decreased overtime. Migratory flow may change this scenario as migrants often come from high HDV endemicity areas. Here, we studied characteristics of HDV infection in Italy, particularly addressed to the birth area of subjects. Chronic HBsAg carriers consecutively referring to 9 units in Italy prospectively enrolled for a six-month period in 2019 were tested for anti-HDV by ELISA. Multiple logistic regression analysis was performed to identify anti-HDV positivity independent predictors. A total of 894 HBsAg-positive subjects were enrolled. Of them, 786 (87.9%) were tested for anti-HDV. Anti-HDV overall prevalence was 9.9% (6.4% in Italian natives and 26.4% in non-natives; P < .001). HDV-RNA was checked in 63 (80.8%) of the 78 anti-HDV+ subjects, and 49 (77.8%) tested positive. Compared to non-natives, Italians were more likely males (male/female 1.6 vs 0.6; P < .05) and older (median age 57 years vs 46 years; P < .05). Multivariate analysis showed that non-natives (OR = 6.02; CI 95% = 3.06-11.84) and cirrhosis (OR 9.6; CI 95% = 5.39-17.30) were independently associated with anti-HDV positivity. A remarkable changing pattern in some characteristics of anti-HDV-positive subjects was observed over 1987-2019: a decreasing male/female ratio, an increasing mean age and proportion of cirrhotic subjects. Anti-HDV prevalence decreased from 7.4% to 6.4% among Italians, increasing from 12.2% to 26.4% among non-natives during 2001-2019. Hence, HDV infection in Italians is further decreasing and mostly affects old people and subjects with advanced disease reflecting a survival effect. Conversely, non-natives are sixfold more likely anti-HDV-positive with an increasing trend. Migratory flow may be a new challenge for HDV infection at the beginning of the third millennium.

Comparison of three therapeutic regimens for genotype-3 hepatitis C virus infection in a large real-life multicentre cohort.

BACKGROUND & AIMS: In the direct-acting antiviral era, treatment of genotype-3 HCV (HCV-GT3) is still challenging. Real-life comparisons between recommended regimens, sofosbuvir (SOF)+daclatasvir (DAC), SOF/velpatasvir (VEL), glecaprevir/pibrentasvir (GLE/PIB), are scarce. We aimed at filling this data gap. METHODS: Sustained virological response 12 weeks after treatment completion (SVR12) was assessed for all HCV-GT3 patients consecutively treated within the Lombardia web-based Navigatore HCV-Network; differences in SVR12 across regimens were evaluated by logistic regression. RESULTS: Of the 2082 subjects with HCV-GT3, 1544 were evaluable for comparisons between regimens: SOF + DAC (1023, 66.2%), SOF/VEL (369, 23.9%), GLE/PIB (152, 9.8%). Patients treated with former regimens were more frequently male, cirrhotic, HIV-positive, pretreated, used ribavirin in their regimen, and had lower baseline HCV-RNA. SVR12 was similar across groups: 94.8% in SOF + DAC, 97.6% in SOF/VEL, 96.7% in GLE/PIB (P = .065). At univariate analysis, SVR12 was associated with female gender (97.9% vs 94.8%, P = .007) and lower median pretreatment Log10 HCV-RNA (5.87 vs 6.20, P = .001). At multivariate logistic regression analysis, treatment with SOF/VEL was associated with a higher likelihood of SVR12 than SOF + DAC, but only in the absence of ribavirin (98% vs 90.3%). Female gender and lower pretreatment HCV-RNA were independently associated with SVR12. CONCLUSIONS: In a large real-life setting of HCV-GT3-infected patients with a high proportion of cirrhosis, the success rate was remarkable. The slight advantage of SOF/VEL on SOF + DAC was significant only without ribavirin. The current prescription shift towards novel regimens (ie SOF/VEL and GLE/PIB) in easier-to-treat patients allows ribavirin-free and shorter schedules without mining SVR12 in this <> genotype.

miR-34a: A Promising Target for Inflammaging and Age-Related Diseases.

The term "inflammaging" describes the chronic, low-grade systemic inflammation that occurs during physiological aging in the absence of an overt infection [...].

When Good Guys Turn Bad: Bone Marrow's and Hematopoietic Stem Cells' Role in the Pathobiology of Diabetic Complications.

Diabetes strongly contributes to the development of cardiovascular disease, the leading cause of mortality and morbidity in these patients. It is widely accepted that hyperglycemia impairs hematopoietic stem/progenitor cell (HSPC) mobilization from the bone marrow (BM) by inducing stem cell niche dysfunction. Moreover, a recent study demonstrated that type 2 diabetic patients are characterized by significant depletion of circulating provascular progenitor cells and increased frequency of inflammatory cells. This unbalance, potentially responsible for the reduction of intrinsic vascular homeostatic capacity and for the establishment of a low-grade inflammatory status, suggests that bone BM-derived HSPCs are not only victims but also active perpetrators in diabetic complications. In this review, we will discuss the most recent literature on the molecular mechanisms underpinning hyperglycemia-mediated BM dysfunction and differentiation abnormality of HSPCs. Moreover, a section will be dedicated to the new glucose-lowering therapies that by specifically targeting the culprits may prevent or treat diabetic complications.

Junctional Adhesion Molecule-C Mediates the Recruitment of Embryonic-Endothelial Progenitor Cells to the Perivascular Niche during Tumor Angiogenesis.

: The homing of Endothelial Progenitor Cells (EPCs) to tumor angiogenic sites has been described as a multistep process, involving adhesion, migration, incorporation and sprouting, for which the underlying molecular and cellular mechanisms are yet to be fully defined. Here, we studied the expression of Junctional Adhesion Molecule-C (JAM-C) by EPCs and its role in EPC homing to tumor angiogenic vessels. For this, we used mouse embryonic-Endothelial Progenitor Cells (e-EPCs), intravital multi-fluorescence microscopy techniques and the dorsal skin-fold chamber model. JAM-C was found to be expressed by e-EPCs and endothelial cells. Blocking JAM-C did not affect adhesion of e-EPCs to endothelial monolayers in vitro but, interestingly, it did reduce their adhesion to tumor endothelium in vivo. The most striking effect of JAM-C blocking was on tube formation on matrigel in vitro and the incorporation and sprouting of e-EPCs to tumor endothelium in vivo. Our results demonstrate that JAM-C mediates e-EPC recruitment to tumor angiogenic sites, i.e., coordinated homing of EPCs to the perivascular niche, where they cluster and interact with tumor blood vessels. This suggests that JAM-C plays a critical role in the process of vascular assembly and may represent a potential therapeutic target to control tumor angiogenesis.

Differences in Mitochondrial Membrane Potential Identify Distinct Populations of Human Cardiac Mesenchymal Progenitor Cells.

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG.

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.